B American Society for Mass Spectrometry, 2012DOI: 10.1007/s13361-012-0452-5

J. Am. Soc. Mass Spectrom. (2012) 23:1670Y1678

RESEARCH ARTICLE

Ambient Mass Spectrometry Imaging:A Comparison of Desorption Ionization by SonicSpray and Electrospray

Christian Janfelt,1 Asger W. Nørgaard2

1Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark2The National Research Centre for the Working Environment, Lersø Parkalle 105, 2100 Copenhagen, Denmark

AbstractEasy ambient sonic spray ionization (EASI) and desorption electrospray ionization (DESI) wereused for imaging of a number of samples, including sections of rat brain and imprints of plantmaterial on porous Teflon. A novel approach termed Displaced Dual-mode Imaging was utilizedfor the direct comparison of the two methods: Images were recorded with the individual rowsalternating between EASI and DESI, yielding a separate image for each technique recordedunder perfectly similar conditions on the same sample. EASI works reliably for imaging of allsamples, but the choice of spray solvent and flow rate is more critical in tissue imaging with EASIthan with DESI. The overall sensitivity of EASI is, in general, slightly lower than that of DESI, andthe representation of the dynamic range is different in images of the two techniques for somesamples. However, for abundant compounds, EASI works well, resulting in images of similarquality as DESI. EASI can thus be used in imaging experiments where the application of highvoltage is impractical or undesirable. The present study is in its nature also a comparison of thecharacteristics of the two techniques, showing results also applicable for non-imaging work, withregards to sensitivity and experimental conditions.

Key words: Easy ambient sonic spray ionization, Desorption electrospray ionization, Imagingmass spectrometry, Ambient mass spectrometry, Displaced dual-mode imaging

Introduction

With the introduction of desorption electrospray ioniza-tion (DESI) in 2004 and direct analysis in real time

(DART) in 2005, it became possible to perform mass-spectrometric analysis directly from surfaces and underambient conditions [1, 2]. The possible applications of thetechniques range from direct analysis of tablets [3, 4] andprofiling of tissues [5] to detection of explosives [6, 7] andanalysis of liquid samples or extracts deposited on surfaces

[8, 9], benefiting from the rapid detection offered by thesedirect techniques.

Since 2004, a variety of related techniques have beeninvented and applied for many different purposes [10–12].As for DESI, several of the techniques are desorptionanalogues of already established spray techniques (e.g.,desorption atmospheric pressure chemical ionization(DAPCI) [6], desorption atmospheric pressure photo ioniza-tion (DAPPI) [13], and nanospray DESI [14, 15], inspired byAPCI, APPI, and nanospray, respectively.

Sonic spray ionization (SSI) [16] is an alternative toelectrospray ionization, using no high voltage and thusrelying solely on the pressure of the nebulizer gas to induceionization. Charged droplets and, consequently, gas-phaseions are formed because of an unbalanced charge distribu-tion during droplet formation in the supersonic spray.Received: 20 April 2012Revised: 28 June 2012Accepted: 17 July 2012Published online: 9 August 2012

Electronic supplementary material The online version of this article(doi:10.1007/s13361-012-0452-5) contains supplementary material, whichis available to authorized users.

Correspondence to: Christian Janfelt; e-mail: cja@farma.ku.dk

Compared to ESI, SSI results in a softer ionization of (e.g.,amino acids) since the ions have lower internal energies thanthose produced in ESI [17, 18]. The desorption analogue ofSSI was presented in 2006 by Haddad and co-workers, firstunder the name desorption sonic spray ionization (DeSSI)[19] and later renamed to easy ambient sonic-sprayionization (EASI) [20]. EASI is one of the simplest of theambient ionization techniques and can be used with anymass spectrometer with an atmospheric pressure inlet. Itdoes not require high voltage or radiation sources and usesonly compressed nitrogen and a spray solvent for ionization.

EASI has proven to be efficient in desorbing and ionizingcompounds from different types of surfaces, such as drugtablets [19], thin layer chromatographic (TLC) plates [21–23],and paper [24–27]. EASI has been utilized to analyze drugstrapped inside the pores of a molecularly imprinted polymer[28] and for real-time studies of the polymerization of organo-functionalized silanes [29] and degradation of drugs [30]. Acombination of DESI-imaging and non-imaging EASI hasbeen suggested for very specific recognition of counterfeitbanknotes [31]. Recently, EASI was used for lipid profiling ofliver tissue, however, only as liver extracts deposited on a papersurface [23] rather than direct analysis of the liver tissue aspreviously presented with DESI [5].

DESI imaging has since its introduction in 2006 [32]been used mainly for tissue analysis, (e.g., in imaging ofdrugs in tissue [33] and examinations of diseased tissue [34–36], but also for imaging of plant material [37, 38], studiesof metabolite exchange between bacteria [39], defencemechanisms of seaweed [40], and analysis of inks [41] andchemical fingerprints [42].

EASI has not been applied for either direct tissue analysisor imaging. In addition, EASI and DESI have never beencompared directly on the same samples. Thus, the scope ofthis study is to explore and compare these two relatedtechniques in a number of imaging experiments of varioussamples. Imaging experiments are well suited for thecomparison of ionization techniques, since the generationof an image inherently provides a pixel-to-pixel comparison,making it easy to distinguish between signal and noise. It isstriking how little signal from a peak is actually needed toprovide a good image. Even small peaks that wouldnormally be dismissed as noise may in the comparison withregions in the image, where the compound is not present,indeed turn out to originate from an actual analyte.Therefore, in comparisons of sensitivity, which is all aboutsignal-to-noise ratios, imaging experiments are well suited,although they of course do not provide any quantitation orlimits of detection.

ExperimentalChemicals

LC-MS grade acetonitrile was purchased from VWRInternational (Herlev, Denmark) and water was prepared

with a Millipore Direct-Q3 UV system (Billerica, MA,USA). All standard compounds were purchased from Sigma-Aldrich Denmark A/S (Copenhagen, Denmark). PorousTeflon (1.5 mm thick, medium pore size of 7 μm, porevolume of 36 %) was purchased from Berghof (Eningen,Germany).

Tissue Sections

The brain was removed from a 9-week old male Sprague-Dawley rat (Taconic, Ry, Denmark), immediately put on dryice for rapid freezing, and subsequently placed in a –80 °Cfreezer. The frozen brain was mounted on a cryotomesample holder under –25 °C with water as the only adhesive.The brain was cut in slices of 14 μm thickness using a LeicaCM3050S cryotome (Leica Microsystems, Wetzlar, Ger-many). Subsequently, the slices were thaw-mounted on glassslides (four sections per glass slide) and stored at –80 °Cuntil time of analysis. On the day of analysis, the sampleslide was taken directly from the freezer to a vacuumdessiccator for 10 min prior to DESI analysis.

Imprints of Plant Material

Plants of St. John’s Wort (Hypericum perforatum) wereharvested in Denmark in September 2011. Imprints weremade by pressing the leaves in a sandwich of porous Teflon,leaf, tissue paper, and silicone rubber (1 mm thick) betweentwo aluminum plates in a vice, as described in further detailselsewhere [38].

Instrumentation

Mass spectrometric data were recorded using a ThermoFisher Scientific LTQ XL Linear Ion Trap Mass Spectrom-eter (San Jose, CA, USA) equipped with a custom-builtDESI imaging ion source. The DESI imaging ion source,described in detail elsewhere [38], was based on a motorizedmicroscope stage from Märzhäuser Wetzlar (Wetzlar, Ger-many) and controlled by an in-house written software. Themass spectra were collected with Xcalibur 2.0 software(Thermo Fisher Scientific) and converted to image files(imzML files) using a conversion tool (www.maldi-msi.org).The images were generated with Data Cube Explorer fromAMOLF, Amsterdam, The Netherlands.

The MS parameters were as follows for negative ionmode experiments: 300 °C heated capillary temperature, –5 kV spray voltage for DESI experiments (0 kV for EASIexperiments), –35 V capillary voltage, –110 V tube lensvoltage, and automatic gain control (AGC) off. In positiveion mode the capillary voltage was 30 V, the tube lensvoltage was 120 V, and the spray voltage was +5 kV forDESI and 0 V for EASI.

For the imaging of rat brain (negative ion mode: scanrange m/z 500–1,000, positive ion mode: scan range m/z600–1,000), the geometric parameters were optimized for

C. Janfelt and A. W. Nørgaard: A Comparison of MS-Imaging by EASI and DESI 1671

EASI, using a nebulizer gas pressure of 10 bar and a 10 μL/min flow of methanol. The ion injection time was 100 ms,and four microscans were averaged for each pixel in theimage. A spatial resolution of 150 μm was used. The DESIimages were recorded under the same conditions, only with±5 kV high voltage applied to the ion source. Imaging of theplant imprint (negative ion mode, scan range m/z 300–700)was performed with a nebulizer gas pressure of 10 bar and a2 μL/min flow of acetonitrile and water (50:50). Theinjection time was 50 ms with six microscans, and thespatial resolution was 100 μm. All images were recorded ina displaced dual-mode imaging approach, as discussedbelow, such that EASI and DESI images were recordedsimultaneously of the same sample. The geometry optimi-zation was always performed for EASI and used for bothionization modes.

The nebulizer gas pressure was tested in the range of 5–15 bar. A major improvement in the EASI signal wasobserved up to 10 bar, above which no further improvementwas observed. For convenience and to avoid unnecessarysmearing of the sample, the nebulizer gas pressure was keptat 10 bar in all experiments reported in this paper.

Results and DiscussionConsiderations on Experimental Conditions

While DESI imaging is typically performed at solvent flowrates of 1–2 μL/min in order to avoid smearing of the sampleleading to degraded spatial resolution, EASI analysis isusually performed a higher flow rates (up to 20 μL/min).DESI is typically performed using mixtures of acetonitrile(or methanol) and water as spray solvent, while mixtures ofmethanol and water or often pure methanol has been usedfor EASI.

EASI imaging was possible using a low solvent flow rate,but for tissue samples the geometry optimization wasdifficult and we were not able to perform any imaging inpositive ion mode. However, we found that the optimumway of optimizing the spray geometry for tissue imagingwith DESI was to turn off the high voltage and thus optimizein EASI mode. Since the spray geometry is very critical inlow flow EASI, the optimal geometry settings could berecognized because of sudden and distinct changes in signal.When the optimal EASI geometry was found, the sprayvoltage was turned back on, resulting in optimal settings forDESI. By increasing the flow rate of the spray solvent(≥5 μL/min) more intense and reliable signals were obtainedfor EASI. Aqueous solvent mixtures at these high flow ratessmear the tissue samples, making imaging impossible.However, by using pure methanol as spray solvent, solventflows up to 20 μL/min were possible without damaging thesample. A flow of 10 μL/min of pure methanol was found tobe ideal for imaging of tissue sections in EASI as well as inDESI. With this, in an imaging context unusually high flowrate, the geometry optimization became very simple for both

EASI and DESI, providing straightforward and reproducibleEASI images of brain tissue in negative as well as in positiveion mode.

The sensitivities of EASI and DESI were tested byanalyzing standard solutions deposited on a porous Teflonsurface, using a 2 μL/min flow of acetonitrile and water(50:50) and a 10 μL/min flow of methanol, respectively.Although 10 μL/min of pure methanol is optimal for EASIanalysis of tissue samples, a 2 μL/min flow of theacetonitrile/water mixture was equally good or better foranalysis of the standards on Teflon. DESI and EASI showedalmost similar sensitivities for compounds, which are easilyionized such as methadone (tens of pg for both techniques).However, for compounds that are more difficult to ionize(e.g., paracetamol) the difference between the two techni-ques is greater in favor of DESI. The details and results ofthese experiments are found in the Supplementary Material.

Displaced Dual-Mode Imaging

In order to make a direct comparison of the two ionizationtechniques, we designed an experiment where the imagingstage was scanning the sample with for example 100 μmspatial resolution in the x-direction but in lanes separated bya 50 μm distance. Every second lane was scanned in EASIand DESI, respectively, thus generating one EASI image andone DESI image, both with 100 μm resolution in bothdirections. In this way, the same sample was imaged withboth techniques, at the same time, and under the sameconditions, thus enabling the best possible comparison of thetwo techniques. In experiments with aqueous (highlyconductive) solvents, an equilibration time of 10 s betweeneach row was sufficient, whereas 50 s were needed betweeneach row for equilibration of the spray when pure methanol(weakly conductive) was used as spray solvent. We term thisapproach displaced dual-mode imaging (DDI) and foresee anumber of other potential applications of it, as discussedbelow.

Imaging of Rat Brain Tissue

EASI and DESI images were recorded in negative andpositive ion mode of sections of a rat brain by DDI.Negative ion mode mass spectra extracted from the twoimages are found in Figure 1, showing EASI and DESIspectra of gray matter areas of the brain (Figure 1a and b) aswell as of white matter areas (Figure 1c and d). The generalobservation is that the spectra, in terms of relative intensities,are similar, showing all the same peaks at quite similarmutual abundances. The only major difference seems to bethat the absolute ion intensities are about two times higherfor DESI than for EASI. In corresponding spectra recordedwith a spray of 2 μL/min ACN/water (70:30), the DESIintensities are about four times higher than the EASIintensities (Figure S1). It is likely that the impact of thehigh voltage is less significant in the experiment with pure

1672 C. Janfelt and A. W. Nørgaard: A Comparison of MS-Imaging by EASI and DESI

methanol as spray solvent because of the low conductivity ofmethanol. The differences between the two techniquesbecome more apparent when images showing the distribu-tions of three different types of lipids are compared(Figure 2). The images differ in the way they show relativeintensities in the spatial dimensions. The two lipids PS(40:6)and PI(18:0/20:4) appear in the DESI images (Figure 2b andd) to be present in significantly higher abundances in thegray matter than in the white matter, while the EASI images(Figure 2a and c) show the lipids in more comparableabundances in the two regions. For the sulfatide ST(24:2)the picture seems opposite with DESI showing the lipidpresent in particular in the white matter but also in parts ofthe gray matter (Figure 2f), while EASI shows a weakerabundance of the lipid in the gray matter areas (Figure 2e).These differences, which are also observed in images madewith a 2 μL/min flow of a 70:30 mixture of acetonitrile/water (Figure S2), are most likely due to the combined effectof the different dynamic ranges of the techniques (asdiscussed below) and their respective vulnerabilities withregards to ion suppression. The less intense signal of ST(24:2) in the gray matter areas in the EASI image inFigure 2e could be ascribed to a greater degree of ionsuppression from the highly abundant phospholipids in thegray matter, due to the fewer charges available in an EASIexperiment.

It is worth noticing that the spatial resolution of the twotechniques is identical, also when compared with images

obtained with a low flow of acetonitrile/water (Figure S2).The high flow of methanol does not cause any smearing ofthe sample.

The positive ion images in Figure 3 show similar effects:the EASI images do not seem to show the same dynamicrange as the DESI images. The difference between the whiteand gray matter areas is observed, but not quite as clearly asin the DESI images. Mass spectra of brain tissue recorded inpositive ion mode are found in the Supplementary Material.

Imaging of Plant Imprints

In another comparison of the two techniques, we performedimaging of Teflon imprints of leaves of Hypericumperforatum, a sample known from a previous study [38].This sample is ideal for this experiment because the porousTeflon surface is well defined and easy to optimize on, andthe plant—studied in hundreds of papers—contains a vastnumber of different compounds.

Figure 4c and d show the EASI and DESI images ofhyperforin (m/z 535.4) in a leaf of H. perforatum. The twoimages are quite similar, in terms of mutual intensities anddetails, spatial resolution etc. Figure 5 shows spectraaveraged over a complete line in each of the images. As inFigure 1 we see significantly higher absolute intensities(factor of 10) with DESI than with EASI. When zooming inon the two spectra (Figure 5c and d), it becomes apparent

Figure 1. Desorption spectra of sections of rat brain recorded in negative ion mode. The upper row, (a), (b) are of the graymatter, and the lower row, (c), (d) are of the white matter. The left column, (a), (c) are recorded with EASI, and the right column,(b), (d) are recorded with DESI. All spectra are extracted from the images in Figure 2

C. Janfelt and A. W. Nørgaard: A Comparison of MS-Imaging by EASI and DESI 1673

Figure 2. Desorption ionization images of rat brain, recorded with 150 μm spatial resolution in negative ion mode. The leftcolumn, (a), (c), (e) are recorded with EASI, and the right column, (b), (d), (f) are recorded with DESI. (a), (b): PS(40:6) (m/z 834.5);(c), (d): PI(18:0/20:4) (m/z 885.5); (e), (f): ST(24:2) (m/z 888.6)

Figure 3. Desorption ionization images of rat brain, recorded with 150 μm spatial resolution in positive ion mode (ionized as K+

adducts). The left column, (a), (c), (e) are recorded with EASI, and the right column, (b), (d), (f) are recorded with DESI. (a), (b): PC(32:0) (m/z 772.5); (c), (d): PC(34:1) (m/z 798.5); (e), (f): PC(36:1) (m/z 826.6)

1674 C. Janfelt and A. W. Nørgaard: A Comparison of MS-Imaging by EASI and DESI

that more of the low-abundance compounds are observedwith DESI than with EASI.

The difference in the dynamic range between EASI andDESI noticed during the imaging of rat brain is also apparentin the images of the plant imprint. Figure 6a and b show theEASI and DESI images of hypericin (m/z 503.1), respec-tively. Hypericin is highly abundant in black glands on theleaf and is therefore transferred in high amounts to theporous Teflon surface during the imprinting process. TheEASI image (Figure 6a) shows three spots of hypericin thatappear to be stretching in lines from the spots toward the

lower right corner of the image. These stretched shapesclearly indicate the movement of the sprayer over the surface(relative movement, as the sprayer is indeed kept still whilethe surface is moving). As the sprayer moves in a line fromleft to right and the lines are scanned one by one, startingfrom the top and moving towards the bottom, apparently thesprayer can, because of the spray solvent and the pressurefrom the nebulizer gas, move to some extent tiny amount ofthe sample in the bottom-right direction. This effect is notobserved in the corresponding DESI image of the very samesurface, and neither were there any visual indications on the

Figure 4. Leaf of Hypericum perforatum. (a) Leaf ; (b) imprint; (c) image of hyperforin (m/z 535.4) recorded by EASI; (d) image ofhyperforin (m/z 535.4) recorded by DESI

Figure 5. Desorption spectra of a leaf of Hypericum perforatum, averaged over one full line from the images in Figure 4. The leftcolumn, (a), (c) are recorded with EASI, and the right column, (b), (d) are recorded with DESI; (c), (d) are the same spectra as (a),(b), but zoomed on the ordinate axis in order to show the signal to noise ratios of the weakest signals

C. Janfelt and A. W. Nørgaard: A Comparison of MS-Imaging by EASI and DESI 1675

surface that the black material on the surface had beenmoved in such lines. It turned out, however, that the lineswere also recorded in the DESI images, although only atlevels so low that they were not visible in the normal colorscaling of the image. If the color scale is changed in order to“zoom in” on lower signal intensities, he lines becomevisible. This is in practice done by defining which absoluteintensity is required to produce a white pixel in the images,and all intensities below this threshold are then properlyscaled in shades of gray such that an intensity of 0 results ina black pixel. Typically, the highest signal in the image ofthe ion being studied is used as threshold. For example, theEASI image in Figure 6a was generated such that anabsolute intensity of 12 (arb. units) equals a white pixel,while the DESI image in Figure 6b with its higher intensitieswas generated with 200 as the threshold. If this threshold inthe DESI image is lowered to 20 (i.e., a factor of 10 lowerthan the highest signal in the image), the hypericin linesshow up in the DESI image as well (Figure 6c). This showsthat the lines in the EASI images are not an indication thatEASI is more sensitive than DESI—the lines are detectedwith both techniques—but that EASI has a somewhat lowerdynamic range than DESI. Although the amount of sampletransported by the sprayer is most likely orders of magnitudelower than in the original hypericin spots (no visual signs ofany transport), the EASI image portrays the concentrationsas being of similar order of magnitude.

The higher sensitivity of DESI observed in the spectra inFigure 5 is also expressed in the images of variouscompounds from the H. perforatum plant from the EASIand DESI experiments. As seen in Figure 6, DESI wascapable of imaging several low abundance compounds.

From the DESI data we could generate images of quercetin(m/z 301.0, Figure 6d) (known to accumulate in the blackglands and therefore have the same distribution as hypericin[43]), an oxidation product of hyperforin (m/z 583.4,Figure 6e) [44] (which therefore has the same distributionas hyperforin), and rutin (m/z 609.2, Figure 6f), which ishomogeneously distributed throughout the leaf surface andalso previously shown to be found in the H. perforatumleaves [45]. None of these compounds showed up in imagesgenerated by EASI.

Imaging of Dyes on Paper and PMMA in PositiveIon Mode

EASI has previously been used for analysis of inks and dyes,and was therefore expected to be suitable for imaging ofsuch samples. Thus, letters written using red and bluepermanent markers on paper and PMMA were used fortesting of the imaging performance of EASI in the positiveion mode. The red pen provides a signal in positive ionmode at m/z 443, corresponding to Rhodamine 6G, and theblue pen provides a signal in positive ion mode at m/z 478,corresponding to Basic Blue 7, as well as a minor signal atm/z 443 due to Rhodamine B. The two isomeric rhodaminescan be distinguished by MS/MS: Rhodamine 6G yields amajor fragment at m/z 415, while the major fragment ofRhodamine B is m/z 399. It should be noted that all threedyes are cationic in their natural state, typically occurring aschloride salts and thus easily ionized. The critical point istherefore extraction and desorption of these compounds fromthe surface.

Figure 6. Desorption ionization images of a leaf of Hypericum perforatum. (a) EASI image of hypericin (m/z 503.1), normalizedsuch that a white pixel corresponds to an intensity of 12 (arb. units). (b) DESI image of hypericin (m/z 503.1), normalized suchthat a white pixel corresponds to an intensity of 200 (arb. units). (c) DESI image of hypericin (m/z 503.1), normalized such that awhite pixel corresponds to an intensity of 20 (arb. units). (d) DESI image of quercetin (m/z 301.0). (e) DESI image of an oxidationproduct of hyperforin (m/z 583.4). (f) DESI image of rutin (m/z 609.2)

1676 C. Janfelt and A. W. Nørgaard: A Comparison of MS-Imaging by EASI and DESI

The imaging performances of EASI and DESI of thethree dyes on paper and PMMA were equally good and withcomparable absolute signal intensities. EASI appears to bequite tolerant with respect to contaminations (e.g., in thesolvent line); the ion of m/z 443 is always the base peak,even on contaminated systems. In comparison, DESI is morevulnerable to contamination and shows several peaks in thebackground on a contaminated system. In EASI with itslower number of charges available, there seems to be a morepronounced preference for donating the limited amount ofcharges to the compound that is the most easily ionized, inthis case the cationic dyes. In case of the blue dye, the m/z443 peak (Rhodamine B) is slightly less prevalent, relativeto Basic Blue 7 at m/z 478, in the EASI spectra than in theDESI spectra. The spatial resolution of the two techniques issimilar, providing very comparable images down to aresolution of about 100 μm. The experimental details andresults of these experiments are found in the SupplementaryMaterial.

ConclusionsWe have shown that EASI can be used for imaging as wellas direct analysis of tissue sections. For analytes such asdyes, the performance is similar to DESI, even presenting alarger tolerance of contaminants in the analytical system.Imaging of tissue samples in negative and positive ionmodes was possible although optimization of the solventcomposition and flow rate is more crucial in EASI than withDESI. A 10 μL/min flow of methanol seems to be optimal,providing images with very high success ratios. Thesesolvent settings also work well for DESI imaging of tissue,demanding very little in terms of geometry optimization inboth positive and negative ion mode. The need for a highersolvent flow rate in EASI is probably due to the lowernumber of charges on the droplets, which is then compen-sated for by generation a larger number of droplets via anincreased flow rate. The EASI images turned out a littledifferently than the corresponding DESI images, indicatingdifferences in the dynamic range or some degree of ionsuppression. However, EASI is well-suited for imaging of avariety of samples and analytes, in particular when theanalyte is of relatively high abundance or easily ionized.EASI imaging can thus prove useful in imaging applicationswhere high voltage (DESI) is impractical or undesirable(e.g., in potential imaging of living organisms).

In conclusion, we have introduced displaced dual-modeimaging, a new method to record two images with differentionization settings of the very same sample simultaneously,by using half the pixel size in the y-direction as in the x-directly and change the ionization settings for every secondrow in the image. We used it here to make a reliableestimation of the impact of the high voltage on thesensitivity in desorption imaging experiment by generatingsimultaneous images with and without high voltage. Thisenabled a direct comparison of the sensitivity and perfor-

mance of EASI and DESI, which is otherwise difficult toobtain because of difficulties in reproducing geometries,samples, and spray conditions. A more general application,however, would be use it as a method to record simultaneousimaging in positive and negative ion modes of the samesample. This would be a good extension of the recent workby Cooks and co-workers, where one sample was used forconsecutive DESI imaging, MALDI imaging, and H&Estaining [46].

AcknowledgmentThe authors thank Niels Wellner for providing the rat brainand Janina Thunig for useful discussions about compoundsin Hypericum perforatum. Support from the CarlsbergFoundation, The Danish Council for Independent Research| Natural Sciences and the Working Environment ResearchFund, Denmark, is gratefully acknowledged.

References1. Takats, Z., Wiseman, J.M., Gologan, B., Cooks, R.G.: Mass spectrom-

etry sampling under ambient conditions with desorption electrosprayionization. Science 306, 471–473 (2004)

2. Cody, R.B., Laramee, J.A., Durst, H.D.: Versatile new ion source forthe analysis of materials in open air under ambient conditions. Anal.Chem. 77, 2297–2302 (2005)

3. Samms, W.C., Jiang, Y.J., Dixon, M.D., Houck, S.S., Mozayani, A.:Analysis of Alprazolam by DART-TOF mass spectrometry in counter-feit and routine drug identification cases. J. Forensic Sci. 56, 993–998(2011)

4. Nyadong, L., Late, S., Green, M.D., Banga, A., Fernández, F.M.: Directquantitation of active ingredients in solid artesunate antimalarials bynoncovalent complex forming reactive desorption electrospray ioniza-tion mass spectrometry. J. Am. Soc. Mass Spectrom. 19, 380–388(2008)

5. Wiseman, J.M., Puolitaival, S.M., Takats, Z., Cooks, R.G., Caprioli,R.M.: Mass spectrometric profiling of intact biological tissue by usingdesorption electrospray ionization. Angew. Chem. Int. Ed. 44, 7094–7097 (2005)

6. Takats, Z., Cotte-Rodriguez, I., Talaty, N., Chen, H., Cooks, R.G.:Direct, trace level detection of explosives on ambient surfaces bydesorption electrospray ionization mass spectrometry. Chem. Commun.1950–1952 (2005)

7. Nilles, J.M., Connell, T.R., Stokes, S.T., Dupont Durst, H.: Explosivesdetection using direct analysis in real time (DART) mass spectrometry.Propellants Explos. Pyrotech. 35, 446–451 (2010)

8. Kauppila, T.J., Talaty, N., Kuuranne, T., Kotiaho, T., Kostiainen, R.,Cooks, R.G.: Rapid analysis of metabolites and drugs of abuse fromurine samples by desorption electrospray ionization-mass spectrometry.Analyst 132, 868–875 (2007)

9. Thunig, J., Flø, L., Pedersen-Bjergaard, S., Hansen, S.H., Janfelt, C.:Liquid-phase microextraction and desorption electrospray ionizationmass spectrometry for identification and quantification of basic drugs inhuman urine. Rapid Commun. Mass Spectrom. 26, 133–140 (2012)

10. Chen, H., Gamez, G., Zenobi, R.: What can we learn from ambientionization techniques? J. Am. Soc. Mass Spectrom. 20, 1947–1963(2009)

11. Alberici, R., Simas, R., Sanvido, G., Romão, W., Lalli, P., Benassi, M.,Cunha, I., Eberlin, M.: Ambient mass spectrometry: bringing MS intothe “real world.” Anal. Bioanal. Chem. 398 (2010)

12. Harris, G.A., Nyadong, L., Fernandez, F.M.: Recent developments inambient ionization techniques for analytical mass spectrometry. Analyst133, 1297–1301 (2008)

13. Haapala, M., Pól, J., Saarela, V., Arvola, V., Kotiaho, T., Ketola, R.A.,Franssila, S., Kauppila, T.J., Kostiainen, R.: Desorption atmosphericpressure photoionization. Anal. Chem. 79, 7867–7872 (2007)

C. Janfelt and A. W. Nørgaard: A Comparison of MS-Imaging by EASI and DESI 1677

14. Roach, P.J., Laskin, J., Laskin, A.: Nanospray desorption electrosprayionization: an ambient method for liquid-extraction surface sampling inmass spectrometry. Analyst 135, 2233–2236 (2010)

15. Laskin, J., Heath, B.S., Roach, P.J., Cazares, L., Semmes, O.J.: Tissueimaging using nanospray desorption electrospray ionization massspectrometry. Anal. Chem. 84, 141–148 (2011)

16. Hirabayashi, A., Sakairi, M., Koizumi, H.: Sonic spray mass spectrom-etry. Anal. Chem. 67, 2878–2882 (1995)

17. Takats, Z., Nanita, S.C., Cooks, R.G., Schlosser, G., Vekey, K.: Aminoacid clusters formed by sonic spray ionization. Anal. Chem. 75, 1514–1523 (2003)

18. Sørensen, M.B., Aaslo, P., Egsgaard, H., Lund, T.: Determination of D/L-amino acids by zero needle voltage electrospray ionisation. RapidCommun. Mass Spectrom. 22, 455–461 (2008)

19. Haddad, R., Sparrapan, R., Eberlin, M.N.: Desorption sonic sprayionization for (high) voltage-free ambient mass spectrometry. RapidCommun. Mass Spectrom. 20, 2901–2905 (2006)

20. Haddad, R., Sparrapan, R., Kotiaho, T., Eberlin, M.N.: Easy ambientsonic-spray ionization-membrane interface mass spectrometry for directanalysis of solution constituents. Anal. Chem. 80, 898–903 (2008)

21. Haddad, R., Milagre, H.M.S., Catharino, R.R., Eberlin, M.N.: Easyambient sonic-spray ionization mass spectrometry combined with thin-layer chromatography. Anal. Chem. 80, 2744–2750 (2008)

22. Eberlin, L.S., Abdelnur, P.V., Passero, A., de Sa, G.F., Daroda, R.J., deSouza, V., Eberlin, M.N.: Analysis of biodiesel and biodiesel-petrodiesel blends by high performance thin layer chromatographycombined with easy ambient sonic-spray ionization mass spectrometry.Analyst 134, 1652–1657 (2009)

23. Alberici, L., Oliveira, H., Catharino, R., Vercesi, A., Eberlin, M.,Alberici, R.: Distinct hepatic lipid profile of hypertriglyceridemic micedetermined by easy ambient sonic-spray ionization mass spectrometry.Anal. Bioanal. Chem. 401, 1655–1663 (2011)

24. Haddad, R., Catharino, R.R., Marques, L.A., Eberlin, M.N.: Perfumefingerprinting by easy ambient sonic-spray ionization mass spectrom-etry: nearly instantaneous typification and counterfeit detection. RapidCommun. Mass Spectrom. 22, 3662–3666 (2008)

25. Abdelnur, P.V., Eberlin, L.S., de Sá, G.F., de Souza, V., Eberlin, M.N.:Single-shot biodiesel analysis: nearly instantaneous typification andquality control solely by ambient mass spectrometry. Anal. Chem. 80,7882–7886 (2008)

26. Simas, R.C., Catharino, R.R., Cunha, I.B.S., Cabral, E.C., Barrera-Arellano, D., Eberlin, M.N., Alberici, R.M.: Instantaneous character-ization of vegetable oils via TAG and FFA profiles by easy ambientsonic-spray ionization mass spectrometry. Analyst 135, 738–744 (2010)

27. Lalli, P.M., Sanvido, G.B., Garcia, J.S., Haddad, R., Cosso, R.G., Maia,D.R.J., Zacca, J.J., Maldaner, A.O., Eberlin, M.N.: Fingerprinting andaging of ink by easy ambient sonic-spray ionization mass spectrometry.Analyst 135, 745–750 (2010)

28. Figueiredo, E.C., Sanvido, G.B., Zezzi Arruda, M.A., Eberlin, M.N.:Molecularly imprinted polymers as analyte sequesters and selective surfacesfor easy ambient sonic-spray ionization. Analyst 135, 726–730 (2010)

29. Nørgaard, A.W., Vaz, B.G., Lauritsen, F.R., Eberlin, M.N.: Real-timemonitoring of the progress of polymerization reactions directly onsurfaces at open atmosphere by ambient mass spectrometry. RapidCommun. Mass Spectrom. 24, 3441–3446 (2010)

30. Amaral, P.H., Fernandes, R., Eberlin, M.N., Höehr, N.F.: Directmonitoring of drug degradation by easy ambient sonic-spray ionizationmass spectrometry: the case of enalapril. J. Mass Spectrom. 46, 1269–1273 (2011)

31. Eberlin, L.S., Haddad, R., Sarabia Neto, R.C., Cosso, R.G., Maia,D.R.J., Maldaner, A.O., Zacca, J.J., Sanvido, G.B., Romao, W., Vaz,

B.G., Ifa, D.R., Dill, A., Cooks, R.G., Eberlin, M.N.: Instantaneouschemical profiles of banknotes by ambient mass spectrometry. Analyst135, 2533–2539 (2010)

32. Wiseman, J.M., Ifa, D.R., Song, Q.Y., Cooks, R.G.: Tissue imaging atatmospheric pressure using desorption electrospray ionization (DESI)mass spectrometry. Angew. Chem. Int. Ed. 45, 7188–7192 (2006)

33. Kertesz, V., Van Berkel, G.J., Vavrek, M., Koeplinger, K.A.,Schneider, B.B., Covey, T.R.: Comparison of drug distribution imagesfrom whole-body thin tissue sections obtained using desorptionelectrospray ionization tandem mass spectrometry and autoradiography.Anal. Chem. 80, 5168–5177 (2008)

34. Eberlin, L.S., Dill, A.L., Costa, A.B., Ifa, D.R., Cheng, L., Masterson,T., Koch, M., Ratliff, T.L., Cooks, R.G.: Cholesterol sulfate imaging inhuman prostate cancer tissue by desorption electrospray ionization massspectrometry. Anal. Chem. 82, 3430–3434 (2010)

35. Masterson, T., Dill, A., Eberlin, L., Mattarozzi, M., Cheng, L., Beck, S.,Bianchi, F., Cooks, R.: Distinctive glycerophospholipid profiles ofhuman seminoma and adjacent normal tissues by desorption electro-spray ionization imaging mass spectrometry. J. Am. Soc. MassSpectrom. 22, 1326–1333 (2011)

36. Janfelt, C., Wellner, N., Leger, P.-L., Kokesch-Himmelreich, J.,Hansen, S.H., Charriaut-Marlangue, C., Hansen, H.S.: Visualizationby mass spectrometry of 2-dimensional changes in rat brain lipids,including N-acylphosphatidylethanolamines, during neonatal brainischemia. FASEB J. 26, 2667–2673 (2012)

37. Li, B., Bjarnholt, N., Hansen, S.H., Janfelt, C.: Characterization ofbarley leaf tissue using direct and indirect desorption electrosprayionization imaging mass spectrometry. J. Mass Spectrom. 46, 1241–1246 (2011)

38. Thunig, J., Hansen, S.H., Janfelt, C.: Analysis of secondary plantmetabolites by indirect desorption electrospray ionization imaging massspectrometry. Anal. Chem. 83, 3256–3259 (2011)

39. Watrous, J., Hendricks, N., Meehan, M., Dorrestein, P.C.: Capturingbacterial metabolic exchange using thin film desorption electrosprayionization-imaging mass spectrometry. Anal. Chem. 82, 1598–1600(2010)

40. Lane, A.L., Nyadong, L., Galhena, A.S., Shearer, T.L., Stout, E.P.,Parry, R.M., Kwasnik, M., Wang, M.D., Hay, M.E., Fernandez, F.M.,Kubanek, J.: Desorption electrospray ionization mass spectrometryreveals surface-mediated antifungal chemical defense of a tropicalseaweed. Proc. Natl. Acad. Sci. U. S. A. 106, 7314–7319 (2009)

41. Ifa, D.R., Gumaelius, L.M., Eberlin, L.S., Manicke, N.E., Cooks, R.G.:Forensic analysis of inks by imaging desorption electrospray ionization(DESI) mass spectrometry. Analyst 132, 461–467 (2007)

42. Ifa, D.R., Manicke, N.E., Dill, A.L., Cooks, G.: Latent fingerprintchemical imaging by mass spectrometry. Science 321, 805 (2008)

43. Holscher, D., Shroff, R., Knop, K., Gottschaldt, M., Crecelius, A.,Schneider, B., Heckel, D.G., Schubert, U.S., Svatos, A.: Matrix-freeUV-laser desorption/ionization (LDI) mass spectrometric imaging at thesingle-cell level: distribution of secondary metabolites of Arabidopsisthaliana and Hypericum species. Plant J. 60, 907–918 (2009)

44. Verotta, L., Appendino, G., Belloro, E., Jakupovic, J., Bombardelli, E.:Furohyperforin, a Prenylated Phloroglucinol from St. John’s Wort(Hypericum perforatum). J. Nat. Prod. 62, 770–772 (1999)

45. Dogrukol-Ak, D., Kirimer, N., Tunçel, M., Aboul-Enein, H.Y.:Determination of rutin in Hypericum perforatum extract by capillaryelectrophoresis. Anal. Lett. 34, 185–191 (2001)

46. Eberlin, L.S., Liu, X., Ferreira, C.R., Santagata, S., Agar, N.Y.R.,Cooks, R.G.: Desorption electrospray ionization then MALDI massspectrometry imaging of lipid and protein distributions in single tissuesections. Anal. Chem. 83, 8366–8371 (2011)

1678 C. Janfelt and A. W. Nørgaard: A Comparison of MS-Imaging by EASI and DESI